Infection of the eukaryotic host cell by Chlamydia trachomatis is initiated by the attachment of elementary bodies (EBs) followed by endocytosis into a membrane-bound vacuole (the inclusion). Expression of early chlamydial gene products (within 2 hours after infection) is required for both the physical transport of the inclusion to the peri-Golgi region and the establishment of fusion competence with sphingomyelin-containing post-Golgi vesicles. This physical interaction with the exocytic pathway has been proposed to be the mechanism whereby the inclusion is isolated from degradation in the lysosomal pathway and constitutes a protected site supporting parasite replication. Knowledge of the molecular responses of the host cell as well as the temporal sequence of bacterial protein expression is crucial to the understanding of the interactions between host and bacterial parasite. Traditionally, this type of protein profiling has been done using radioactive metabolic labeling followed by comparative 2D gel technology. This method is not robust due to limitations in sensitivity, quantita-tion, and detection of post-translational modifications. A major aim of this project is to develop methods using mass spectrometry to rapidly profile differentially expressed proteins and their post-translational modifications in total cell lysate mixtures. These methods will be used to monitor the changes in protein expression and post-translational modifications of the host cell in response to chlamydial entry, establishment of the inclusion, and subsequent fusion events at the peri-Golgi region. Additionally, bacterial protein expression will be temporally profiled during these time points. These novel proteomic approaches will identify key molecular players for subsequent molecular dissection to determine functional interactions between host and parasite. [unreadable] [unreadable]